Biomedical subjects

D W Hedley

Publications and source records attributed to D W Hedley.

At least 37 records · Page 2Linked to original sources

PubMed2000

Microregional heterogeneity of non-protein thiols in cervical carcinomas assessed by combined use of HPLC and fluorescence image analysis.

Under low oxygen conditions, non-protein thiols (NPSHs, non-protein sulfhydryls) can effectively compete for DNA radicals sites and hence represent a potentially important cause of radiation resistance in the clinic. Intra- and intertumoral heterogeneity of glutathione (GSH) and cysteine were assessed in cryostat sections of multiple biopsies obtained from 10 cervical carcinomas by the combined use of a sensitive high-performance liquid chromatography (HPLC) method and a fluorescence image analysis technique to examine the spatial distribution of NPSHs in tumor tissue. Glutathione concentrations ranged from 1.98 to 4.42 mM; significant (> or =1 mM) concentrations of cysteine, a more effective radioprotector than GSH, were found in some tumors. By HPLC, the intratumoral heterogeneity of NPSHs was relatively small compared with the intertumoral heterogeneity. The histochemical stain 1-(4-chloromercuryphenoylazo)-2-napthol (mercury orange), which binds to GSH and cysteine, was used to determine the spatial distribution of NPSHs in tumor tissue. A comparison of NPSH levels in serial cryostat sections showed a close correlation between NPSH values determined by HPLC and mercury orange fluorescence quantification. Using fluorescence image analysis, an approximately 2-fold increase of NPSHs in tumor versus nonmalignant tissue was observed in the same section. Because some cervical carcinomas contain radiobiologically important levels of cysteine, agents that target the biochemical pathways maintaining tumor cysteine have therapeutic potential as adjuncts to radiotherapy in cervix cancer patients.

Adenocarcinoma↗

PubMed1999

Bcl-2 targeted to the endoplasmic reticulum can inhibit apoptosis induced by Myc but not etoposide in Rat-1 fibroblasts.

Bcl-2 is a key inhibitor of a broad range of apoptotic pathways, yet neither the mechanism of action nor the role of Bcl-2 subcellular localization are well understood. The subcellular localization of Bcl-2 includes the mitochondrial membrane as well as the contiguous membrane of the endoplasmic reticulum and nuclear envelope. Most studies suggest that the ability of Bcl-2 to confer cell survival is dependent upon its localization to the mitochondria. In this manuscript, we show that Bcl-2 targeted to the endoplasmic reticulum can inhibit Myc-, but not etoposide-induced apoptosis in the Rat-1 fibroblast cell line. By contrast, wild type Bcl-2 can inhibit apoptosis triggered by either death agonist. We further show both Myc and etoposide trigger disruption of mitochondrial membrane potential (MMP) and induce poly-ADP ribose polymerase (PARP) cleavage, but release of calcium was not evident. Bcl-2 abrogates apoptosis at or upstream of MMP depletion showing that Bcl-2 does not have to reside at the mitochondria to prevent apoptosis. These results further elucidate the biochemical events associated with Myc- and etoposide-induced apoptosis and significantly advance our understanding of Bcl-2 function.

Animals↗

PubMed1999

Increased sensitivity of acute myeloid leukemias to lovastatin-induced apoptosis: A potential therapeutic approach.

We recently demonstrated that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of de novo cholesterol synthesis, was a potential mediator of the biological effects of retinoic acid on human neuroblastoma cells. The HMG-CoA reductase inhibitor, lovastatin, which is used extensively in the treatment of hypercholesterolemia, induced a potent apoptotic response in human neuroblastoma cells. This apoptotic response was triggered at lower concentrations and occurred more rapidly than had been previously reported in other tumor-derived cell lines, including breast and prostate carcinomas. Because of the increased sensitivity of neuroblastoma cells to lovastatin-induced apoptosis, we examined the effect of this agent on a variety of tumor cells, including leukemic cell lines and primary patient samples. Based on a variety of cytotoxicity and apoptosis assays, the 6 acute lymphocytic leukemia cell lines tested displayed a weak apoptotic response to lovastatin. In contrast, the majority of the acute myeloid leukemic cell lines (6/7) and primary cell cultures (13/22) showed significant sensitivity to lovastatin-induced apoptosis, similar to the neuroblastoma cell response. Of significance, in the acute myeloid leukemia, but not the acute lymphocytic leukemia cell lines, lovastatin-induced cytotoxicity was pronounced even at the physiological relevant concentrations of this agent. Therefore, our study suggests the evaluation of HMG-CoA reductase inhibitors as a therapeutic approach in the treatment of acute myeloid leukemia.

Acute Disease↗

PubMed1999

p53 gene status and chemosensitivity of childhood acute lymphoblastic leukemia cells to adriamycin.

The role of p53 as a determinant of sensitivity of ten childhood acute lymphoblastic leukemia (ALL) cell lines to Adriamycin (ADR) was investigated. ADR-sensitive cell lines were found to have wild-type (wt) p53, whereas resistant cell lines contained point mutations in the gene. The basal level of wt p53 protein in sensitive cells was lower than that of mutant p53 in resistant cells, however, after ADR treatment a 6- to 20-fold dose-dependent increase in wt p53 was observed, whereas mutant p53 increased only twofold. The percentage of apoptotic cells in ADR-sensitive lines with wt p53 ranged from 43 to 93% following ADR treatment, whereas that in resistant lines with mutant p53 was only 8-13%. The ratio of constitutive levels of Bax/Bcl-2 was significantly higher in cells containing wt p53 than in cells with mutant p53. These results suggest that p53 gene status and the ability of p53 to induce apoptosis may be determinants of sensitivity to ADR in childhood ALL cells.

Antineoplastic Agents↗

PubMed1999

Association between tissue hypoxia and elevated non-protein sulphydryl concentrations in human cervical carcinoma xenografts.

A double staining technique was developed for the simultaneous measurement of tissue hypoxia and the concentration of non-protein sulphydryls (NPSH), based on the fluorinated nitroimidazole EF5 and the fluorescent histochemical NPSH stain 1-(4-chloromercuriphenoylazo)-naphthol-2 (mercury orange). Cryostat sections of tumour tissue were examined by fluorescence image analysis, using a computer-controlled microscope stage to generate large tiled field images of the cut tumour surface. This method was applied to the human cervical squamous cell carcinoma lines ME180 and SiHa, grown as xenografts in severe combined immunodeficient (SCID) mice, in order to determine if there is a systematic relationship between tissue hypoxia and NPSH levels. Hypoxic regions of the tumours, defined by EF5 labelling, were found to show greater NPSH concentrations relative to better oxygenated regions. This is probably due to increases in glutathione, since the ME180 and SiHa xenografts contained low levels of cysteine and metallothionein; the other major cellular thiols that can bind to mercury orange. Because the effects of glutathione on radiation and chemotherapy resistance are likely to be greater under hypoxic conditions, these results have potentially important implications for the study of resistance mechanisms in solid tumours.

Animals↗

PubMed1998

Antileukemic action of buthionine sulfoximine: evidence for an intrinsic death mechanism based on oxidative stress.

The glutathione-depleting agent buthionine sulfoximine (BSO) was found to be toxic to some AML blast populations. This toxicity was manifested as the appearance of high levels of reactive oxygen generation in GSH-depleted cells, and later by the loss of mitochondrial membrane potential and an increase in intracellular calcium. Striking heterogeneity in BSO sensitivity was observed in a series of four human AML cell lines, and in fresh leukemic blasts obtained from eight AML patients. In some cases, toxicity was seen at BSO concentrations as low as 1 microM; approximately 100-fold less than the plasma levels achieved in patients treated with BSO as a drug resistance reversing agent. Based on these results we propose that some AML blast populations are unusually dependent on GSH-based antioxidant mechanisms, due to high intrinsic rates of reactive oxygen generation. The mitochondrial respiratory chain is the most likely source of this reactive oxygen. Because toxicity is seen at clinically achievable concentrations of BSO, this agent might have antileukemic activity in patients.

Adult↗

PubMed1998

An oxidative stress-mediated death pathway in irradiated human leukemia cells mapped using multilaser flow cytometry.

OCI/AML-2 acute myeloid leukemia cells were found to undergo apoptosis after treatment with y rays from a 137Cs source. Multilaser flow cytometry techniques using probes for live cell function were used to monitor the biochemical changes that occurred prior to the loss of surface membrane integrity. These showed increases in the generation of reactive oxygen species (ROS) and in the glutathione (GSH) content of irradiated cells. An additional population of cells that showed a further increase in ROS and depletion of GSH was seen in irradiated cells but not in controls. This population showed loss of mitochondrial membrane potential (deltapsim), indicative of the mitochondrial permeability transition, and exposure of phosphatidylserine on the cell surface. Increases in intracellular calcium were observed in a proportion of these low-deltapsi(m)/high-ROS cells. Similar findings were seen using the antileukemia drug cytosine arabinoside (ara-C), although cell cycle analysis showed that the loss of deltapsi(m) occurred mainly in G1 phase with ara-C treatment, and mainly in G2 phase with irradiation. Furthermore, the protective effect of overexpression of BCL2 was more pronounced after ara-C treatment than with radiation. Cells of the TP53 (formerly known as p53)-null human AML line OCI M2 showed growth arrest in G2 phase after radiation treatment, with no loss of deltapsi(m) or morphological changes indicative of apoptosis. The flavine-dependent oxidoreductase inhibitor diphenylene iodonium failed to inhibit generation of ROS in irradiated OCI/AML-2 cells, indicating that the mechanism is unlikely to involve the TP53-induced gene PIG3. These results show that oxidative stress can occur in irradiated human leukemia "blasts", and may play a direct role in radiation-induced apoptosis.

Antimetabolites, Antineoplastic↗

PubMed1997

Relationships between the mitochondrial permeability transition and oxidative stress during ara-C toxicity.

The mitochondrial permeability transition and oxidative stress seem to be critical alterations in cellular physiology that take place during programmed cell death. Failure to undergo apoptosis is associated with drug resistance in acute myeloid leukemia and other cancers. Therefore, it is important to establish causal relationships between the physiological changes that take place in apoptosis, because these are potential targets for novel treatment strategies to overcome this form of drug resistance. We describe the use of multilaser flow cytometry methods to make correlated measurements of mitochondrial membrane potential (MMP), the generation of reactive oxygen intermediates, the cellular content of reduced glutathione (GSH), intracellular calcium, and exposure of phosphatidylserine on the cell surface. Using these combined methods, we have mapped a "death sequence" that occurs after treatment of leukemic blasts with clinically relevant concentrations of 1-beta-D-arabinofuranosylcytosine (ara-C). Dual labeling of MMP and cellular glutathione content showed that loss of MMP, indicative of the permeability transition, took place in cells that were depleted of glutathione. The loss of MMP coincided with phosphatidylserine exposure and preceded a state of high reactive oxygen generation. Finally, there was an increase in intracellular calcium. These results demonstrate that the mitochondrial permeability transition takes place during ara-C toxicity but suggest that this occurs downstream of the loss of GSH. Thus, oxidative stress after ara-C-induced toxicity seems to be a biphasic phenomenon, with the permeability transition occurring after a depletion of GSH and preceding a state of high reactive oxygen generation.

Calcium↗

PubMed1997

Feasibility of obtaining breast epithelial cells from healthy women for studies of cellular proliferation.

Increased dietary fat intake and rate of breast epithelial cell proliferation have each been associated with the development of breast cancer. The goal of this study was to measure the effect of a low fat, high carbohydrate diet on the rate of breast epithelial cell proliferation in women at high risk for breast cancer. Women were recruited from the intervention and control groups of a randomized low fat dietary intervention trial, breast epithelial cells were obtained by fine needle aspiration, and cell proliferation was assessed in these samples using immunofluorescent detection of Ki-67 and PCNA. The effects of needle size and study group on cell yield and cytologic features of the cells were also examined. Fifty three women (20 in the intervention group and 33 in the control group) underwent the biopsy procedure. Slides from 38 subjects were stained for Ki-67 and from 14 subjects for PCNA. No cell proliferation (fluorescence) was detected for either Ki-67 or PCNA in any of the slides. Epithelial cell yield and number of stromal fragments were greater with a larger needle size. Numbers of stromal fragments and bipolar naked nuclei were greater in the low fat as compared to the control group but no differences in epithelial cell yield were observed between the two groups. This study confirms that fine needle aspiration biopsy is a feasible method of obtaining epithelial cells from women without discrete breast masses, but suggests that cell proliferation cannot be assessed using Ki-67 and PCNA in such samples.

Biomarkers↗

PubMed1997

A novel energy dependent mechanism reducing daunorubicin accumulation in acute myeloid leukemia.

Using cyclosporin A (CsA) to inhibit P-glycoprotein (P-gp) function we showed previously that there was a discordance between the ability of acute myeloid leukemic (AML) blast cells to accumulate daunorubicin and P-gp antigen expression (Xie et al, Leukemia 1995; 9:1882-1887). This discordance suggests that a CsA-sensitive drug efflux mechanism distinct from P-gp is expressed in many clinical samples. In the present study using the ATP depleting agents cyanide, azide, or dinitrophenol to inhibit energy dependent transport processes, we observed even larger increases in daunorubicin accumulation than were seen with CsA. Similar patterns were seen in a wide range of P-gp negative human cancer cell lines. Also the observed cyanide effect did not correlate with the expression of mRNA for multidrug resistance-associated protein (MRP), the only other member of the ABC family of membrane transporters that is known to be capable of effluxing daunorubicin. Thse results suggest that daunorubicin accumulation in many cases of AML is modulated by one or more novel energy-dependent processes that are distinct from P-gp or MRP. We speculate that this novel drug transport mechanism(s) may influence the response of AML patients to daunorubicin and other therapeutic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Effects of topoisomerase I inhibition on the expression of topoisomerase II alpha measured with fluorescence image cytometry.

A fluorescence image cytometry technique was developed to measure the effects of topotecan, a topoisomerase I inhibitor, on the nuclear expression of topoisomerase II alpha in a series of patients with refractory or relapsed acute myeloid leukemia (AML). We used a commercially available affinity-purified rabbit polyclonal antibody and a fluorescein-conjugated secondary antibody. By using DAPI as a DNA counterstain and dual wavelength excitation, it was possible to measure enzyme expression in the cell nucleus, and to examine its cell cycle phase distribution. In human acute leukemia cell lines, topoisomerase II alpha expression was greatest in late S and G2 phases, but in leukemia patient samples the enzyme expression appeared to be much less cell cycle dependent. There was considerable interpatient variation in the effects of topotecan on topoisomerase II alpha expression in the leukemia patients, with a threefold increase in the median value after 48 h followed by a decline to pretreatment levels after 5 days of treatment with the topoisomerase I inhibitor. Although these findings should be treated with caution because of the small number of cases studied, they support the prediction that topoisomerase I inhibitors might be capable of increasing sensitivity to topoisomerase II active drugs such as anthracyclines and epipodophyllotoxins by upregulating topoisomerase II expression. They also illustrate the potential value of fluorescence image cytometry for making sequential measurements of the effects of drug resistance modulating agents in cancer patients.

Animals↗

PubMed1996

Generation of reactive oxygen intermediates after treatment of blasts of acute myeloblastic leukemia with cytosine arabinoside: role of bcl-2.

Cytosine arabinoside is usually considered to be lethal by incorporation into DNA followed by chain termination. Recently, we have reported that the radical scavenger N-acetyl-cysteine (NAC) protects cultured clonogenic AML blast cells from the lethal affects of Ara-C if given before the drug. This observation provides indirect evidence that toxic reactive oxygen intermediates (ROI) are generated in AML blast cells following Ara-C-induced damage to DNA. In the present paper we present evidence in support of this hypothesis. Using flow cytometry and multiple fluorescent probes for live cell function, we have mapped a sequence of discrete stages that occur during Ara-C cytotoxicity. An early event was the increased generation of ROI. Initially this oxidative stress was countered by an increase in the cellular content of reduced glutathione (GSH), but cells then underwent an abrupt transition to a state characterized by low GSH and very high ROI generation indicative of collapse of cellular redox balance. Next, the capacity to maintain low intracellular ionized calcium was lost, probably due to lipid peroxidation at membrane sites of calcium regulation. Finally, surface membrane integrity was lost. Concurrent measurements of clonogenic cell survival insured the relevance of these flow cytometry measurements to the stem cell population. We used OCI/AML-2 cells transfected with bcl-2 to look for the place in this sequence where bcl-2 protein protects cells against apoptosis; bcl-2 transfectants showed an increase in ROI generation similar to controls, but were able to maintain GSH levels in the face of this oxidative stress. We conclude that oxidative stress plays a major role in Ara-C toxicity, and that bcl-2 protein protects cells by maintaining cellular redox balance in a reducing state. These studies complement previous work showing how regulators of AML growth affect the sensitivity of blast cells to Ara-C by changing the concentration or stability of bcl-2 protein.

Antimetabolites, Antineoplastic↗

PubMed1995

Biochemical modulation of iododeoxyuridine by N6-[4-(morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[cd]indole glucuronate (AG-331) leading to enhanced cytotoxicity.

Inhibition of thymidylate synthase (TS) may increase incorporation of thymidine analogues into DNA, leading to increased inhibition of colony formation in tumor cells. We have reported previously that TS inhibition by N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6,-ylmethyl)-N -methylamino]-2 - thenoyl)-L-glutamic acid (ICI D1694 or Tomudex), a folate-based TS inhibitor, increases the cytotoxicity of iododeoxyuridine (IdUrd), a thymidine analogue, in MGH-U1 human bladder and HCT-8 human colon cancer cells. N6-[4-(Morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]-indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. Exposure of MGH-U1 cells to 5 microM AG-331 for 24 hr decreased clonogenic survival by 30%, but almost completely inhibited TS activity. IdUrd is a cytotoxic thymidine analogue, with IC50 and IC90 values after 24-hr exposures in MGH-U1 cells of 13 and 81 microM, respectively. The combination of IdUrd and AG-331 resulted in an enhanced antitumor effect, as compared with the effect of either agent alone. The cytotoxic IC50 of IdUrd decreased from 13 to 1.5 microM, and the IC90 decreased from 81 to 5 microM with the addition of 5 microM AG-331. Biochemical studies of the combination revealed that pretreating MGH-U1 cells with 5 microM AG-331 increased IdUrd incorporation into cellular DNA by 3.8-fold. This increased incorporation was associated with a greater proportion of DNA single-strand breaks than observed with either agent alone, and the combination of 5 microM AG-331 plus IdUrd produced up to a 2.5-fold increase in DNA single-strand breaks as compared with IdUrd alone. The effects of AG-331, IdUrd, and the combination of IdUrd and AG-331 on the colony-forming ability of normal human bone marrow CFU-GM cells was determined as a measure of myelosuppression. The combination of IdUrd and AG-331, at the same concentrations as those used in the MGH-U1 cells, produced a wider therapeutic index relative to that of IdUrd alone, and the therapeutic index for the combination was 6.5, as compared with 4.0 for IdUrd plus ICI D1694 in previous studies from this laboratory. These observations suggest that the combination of IdUrd and AG-331 may enhance antitumor effects with minimal myelosuppression in vivo.

Antimetabolites, Antineoplastic↗

PubMed1995

Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression.

The effects of de novo dTMP inhibition by N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2- thenoyl)-L-glutamic acid (D1694) or N6-[4-(morpholinosulfonyl)benz]-N6-diaminobenz[cd]indole glucuronate (AG-331) on clonogenic survival, thymidylate synthase (TS) and thymidine kinase (TK) activity, and expression of S-(p-nitrobenzyl)-6-thioinosine-sensitive nucleoside transporter (NT) sites were addressed in the human bladder cancer cell line, MGH-U1. These two TS inhibitors are structurally diverse. D1694 is a folate-based TS inhibitor, whereas AG-331 is a novel agent that inhibits the cofactor binding site of the enzyme. They also differ with respect to their cytotoxic effects in this cell line; D1694 cytotoxic 50% inhibitory concentration (IC50) and IC90 were 6.0 and 9.0 nM, respectively and IC50 and IC90 for TS inhibition were 2.5 and 4.8 nM, respectively. In contrast, AG-331 cytotoxic IC50 could not be achieved even at concentrations of up to 20 microM for 24-h exposures, and IC50 and IC90 for TS inhibition were 0.7 and 3.0 microM, respectively. Similar effects for D1694 and AG-331 were observed in their modulation of TK activity and NT expression. 5-(SAENTA-x8)-Fluorescein, a highly modified form of adenosine incorporating a fluorescein molecule which binds with a 1:1 stoichiometry to S-(p-nitrobenzyl)-6-thioinosine-sensitive NT sites, was used to investigate the expression of NT following exposure of cells to D1694 and AG-331. TK activity was addressed by the metabolism of [3H]thymidine to [3H]TMP by cellular extracted protein and by an alternative flow cytometric method using a modified form of thymidine incorporating a fluorescent molecule, dansyl-5-amino-2-deoxyuridine. Results obtained by both methods were comparable. At concentrations of 5 and 10 nM, D1694 increased TK activity 2.3-4.5-fold and NT expression 34-39-fold. AG-331, at concentrations of 5 and 10 microM, increased TK activity 1.8-2.5-fold and NT expression 22-31-fold, respectively. These data suggest that TK activity and NT expression have a common regulatory mechanism which is sensitive to endogenous dTTP pools and that the salvage pathway is a complex system of kinases coordinated with transport of nucleosides.

Carrier Proteins↗

PubMed1995

Relationship of DNA topoisomerase II alpha and beta expression to cytotoxicity of antineoplastic agents in human acute lymphoblastic leukemia cell lines.

The levels of expression of topoisomerase II alpha and topoisomerase II beta were investigated in six established cell lines of human childhood acute lymphoblastic leukemia (ALL) as a function of doubling time, cell cycle distribution, and of sensitivity to the antineoplastic agents Adriamycin and etoposide. The slowest growing cell line, ALL-G, was most sensitive to both drugs, whereas the fastest growing cell line, ALL-C, was 15.3- and 6.4-fold more resistant than ALL-G to Adriamycin and etoposide, respectively. Furthermore, ALL-W, the second most rapidly dividing cell line, was most resistant to both Adriamycin (22.8-fold) and etoposide (14.1-fold). Expression of topoisomerase II alpha varied inversely with doubling time, whereas no correlation was found between topoisomerase II beta levels and doubling time. Expression of topoisomerase II beta varied inversely with that of topoisomerase II alpha. The level of topoisomerase II alpha correlated directly with the percentage of cells in S and G2-M phases, whereas topoisomerase II beta expression varied directly with the number of cells in G1. An inverse correlation was found between the level of expression of topoisomerase II beta and resistance to Adriamycin, whereas a direct correlation was observed between the level of expression of topoisomerase II alpha and resistance to Adriamycin. Studies with etoposide, although not statistically significant, were consistent with the pattern observed with Adriamycin. These findings suggest that in ALL cells, cytocidal activity of Adriamycin and etoposide may be mediated, at least in part, by topoisomerase II beta.

Antineoplastic Agents↗

PubMed1995

Differential effects of depleting agents on cytoplasmic and nuclear non-protein sulphydryls: a fluorescence image cytometry study.

The intracellular distribution of glutathione (GSH) was measured by a quantitative image cytometry method, using the sulphydryl-reactive agent mercury orange. This readily forms fluorescent adducts with GSH and other non-protein sulphydryls (NPSH), but reacts much more slowly with protein sulphydryls. Under optimum staining conditions mean integrated mercury orange fluorescence per cell was closely correlated with a standard biochemical assay for GSH. Use of the DNA dye DAPI as a counterstain allowed measurement of nuclear NPSH. The mean nuclear-cytoplasmic ratio was 0.57 +/- 0.05. Isolation of nuclei under aqueous conditions resulted in the loss of approximately 90% of mercury orange fluorescence, compared with nuclear fluorescence from intact cells, suggesting that background labelling of protein sulphydryls or other macromolecules is low. Depletion of GSH with N-ethylmaleimide or diethylmaleate decreased mercury orange fluorescence in the nucleus and cytoplasm to a similar extent. In contrast, mercury orange fluorescence in the nucleus was much more resistant to DL-buthionine-S,R-sulphoximine (BSO) depletion than that in the cytoplasm. This finding is compatible with a distinct pool of GSH in the nucleus that is comparatively resistant to BSO depletion. Alternatively, the retention of fluorescence in the nucleus following GSH depletion by BSO treatment might be due to accumulation of cysteine. These findings have implications for cancer treatment since the level of NPSH in the nucleus might be a more important determinant of resistance to DNA-damaging agents than that in cytoplasm. The image cytometry method described here is quantitative, allows a measure of tumour cell heterogeneity and can be applied to small biopsy samples obtained by fine-needle aspiration. Thus it appears suitable for prospective clinical studies in cancer patients, and for monitoring the effects of GSH-depleting agents used as adjuncts to cancer chemotherapy or radiotherapy.

Animals↗

PubMed1995

Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis.

Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-x8)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds. with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-x8)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools.

Antimetabolites↗

PubMed1995

Discordant P-glycoprotein antigen expression and transport function in acute myeloid leukemia.

Expression of the multidrug resistance efflux pump P-glycoprotein (Pgp) was measured in a series of AML patients using two flow cytometry methods. Transport function was assessed by measuring the modulating effect of the Pgp inhibitor cyclosporin A (CsA) on the cellular accumulation of daunorubicin, and Pgp antigen expression by surface immunofluorescence using the MRK-16 antibody. Both methods showed a wide range of values for Pgp expression between individual patients, but in contrast to a series of cell lines expressing Pgp there was no correlation between antigen expression and transport function in the clinical samples. As previously reported for chronic lymphocytic leukemia (CLL), pretreatment with neuraminidase markedly improved MRK-16 staining in some cases, indicating that abnormal glycosylation can cause epitope masking in AML blasts. Because experience with cell lines shows that Pgp expression is a continuous variable which correlates with the level of drug resistance, rather than the 'positive' or 'negative' which are frequently reported by clinical flow cytometry laboratories, we used a calibration procedure to estimate the actual number of Pgp molecules expressed in the AML samples. Despite the additional refinements of neuraminidase treatment and antigen quantification, the correlation between Pgp antigen expression and daunorubicin accumulation remained extremely weak (r = 0.11; P = 0.63). It is suggested that the assay for transport function can detect molecules that affect daunorubicin accumulation but are antigenically distinct from classical P-glycoprotein. Heterogeneity of multidrug resistance efflux pumps might in part explain the relatively weak prognostic significance of immunofluorescence detection of Pgp in AML patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗